The present invention relates to an assay method and particularly to an assay method for catheter related sepsis (CRS) in indwelling catheters. Although the invention is described largely relative to blood, the invention is also applicable to any body fluid such as lymph, or material removed from the catheter wall such as fibrin.
Catheter-related sepsis (CRS) is a major problem when patients are fed intravenously via a central vein. Diagnosis is based upon removal of the catheter and culture of the catheter tip. However, the majority of catheters removed on suspicion of sepsis are found to be sterile, and results of tip-cultures are often unavailable for 48 hours after removal of the catheter. There is therefore a need for a rapid and reliable test to confirm whether or not a catheter is colonised.
The acridine orange leucocyte cytospin (AOLC) test has been shown to be a sensitive test in the diagnosis of CRS in neonates, with a result in under one hour. The test has not previously been effective in adults because of the more extensive of dilution of organisms in the greater blood volume present in adults. The success of the AOLC test is dependent upon the direct detection of bacteria from a small sample (50 xcexcl) of blood aspirated from the catheter, using ultraviolet microscopy. The high sensitivity is now thought to be due to the large concentration of bacteraemia per milliliter of blood in neonates with catheter-related sepsis. Since the relative quantitative level of bacteraemia in adults is lower, the test is not sensitive enough. When an indwelling catheter is inserted into a vein and retained in situ, a fibrin layer forms on the catheter lumen surface particularly adjacent to the tip. This fibrin is adhesive for bacteria which over time will colonise the lumen walls to create CRS. Since the bacteria are fixed, aspiration of a body fluid such as blood or lymph will not dislodge the bacteria from the walls and accordingly a technique is required to release more bacteria from the catheter into the lumen of the catheter. An endoluminal brush (Daymark Medical Industries Inc, WO94/25620) may be used for this purpose. The brush comprises nylon bristles wound tightly round the distal end of the stainless steel wire. The brush is inserted through the hub of the catheter and passed the full length of the catheter until a loss of resistance is felt as the brush exits the distal end of the catheter. The bristles are designed to remove fibrin, debris and bacteria from the surface of the catheter and hence allow detection of sepsis without removal of the catheter.
Thus, there is a release of debris and bacteria into the lumen from the surface of the catheter. The fibrin, bacteria and/or bacterial debris may then be aspirated when a body fluid (eg blood) sample is taken from the catheter. As we show below, the use of an endoluminal brush or similar means increases the quantitative level of bacteraemia in the aspirated sample from an infected catheter, so as to reach the sensitivity level of the AOLC and similar tests. Alternatively or additionally, fibrin attached to the brush may also be examined by suitable methods.
According to a first feature therefore of the present invention there is provided a method of assaying biologically active material from an in situ catheter lumen for the presence of fibrin, a microorganism and/or debris thereof which comprises subjecting the biologically active material from the in situ catheter lumen to analysis;
characterised in that the lumen is subjected to mechanica action to dislodge the biologically active material from the wall of the catheter lumen in an amount sufficient to allow identification of target microorganisms without culturing. This mechanical action may be effected by means of an endoluminal brush. A particularly effective test for use in this method is a leucocyte cytospin technique or a rapid antigen test. The dislodged biologically active material may be obtained by immediate aspiration of a body fluid, such as blood, after mechanical action, or the means of applying mechanical action itself may be assayed for the presence of the biologically active material.
In the present invention we have described the use of an acridine orange leucocyte cytospin technique. However, it will be appreciated that the invention will improve the sensitivity of most appropriate assays.
The method may be assisted by concentration of the fibrin, microorganism, bacteria and bacterial debris after aspiration. This may be effected by mechanical means such as filtration or centrifugation, especially differential centrifugation at different speeds in order to separate solid components from a supernatant. The assay may be performed on the concentrated solids or may, using a rapid antigen test, be performed on the supernatant or the solid components.
Suitable staining agents may be acridine orange or Hoechst stains for example. The rapid antigen test may be effected by using one or more antibodies to give a positive response to one or more selected microorganisms.
A single assay kit may therefore include a mixed plurality of antibodies responsive to selected target microorganisms. These may include for example:
coagulase negative staphlycoccus;
Staphylococcus aureus; 
Escherichia coli; 
Pseudomonas aeruginosa; 
Streptococcus pneumoniae; and
Candida albinicans. 
In a further aspect of the present invention, there is provided an analysis kit for the direct detection of a target microorganism in a catheter lumen, said kit comprising:
a) assay means for the direct detection of a microorganism in an uncultured sample; and
b) means to induce mechanical action within the catheter lumen to dislodge fibrin, the microorganism and/or debris thereof adherent thereto.
The assay means may comprise means for effecting an AOLC or a Hoechst staining test or a rapid antigen test, and the mechanical means to induce mechanical action may be an endoluminal brush.
The invention will now be described by way of illustration only with reference to the following clinical study: